CN101082569A - Method for measuring amino acid concentration and amino acid diagnose reagent kit - Google Patents
Method for measuring amino acid concentration and amino acid diagnose reagent kit Download PDFInfo
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- CN101082569A CN101082569A CN 200710024763 CN200710024763A CN101082569A CN 101082569 A CN101082569 A CN 101082569A CN 200710024763 CN200710024763 CN 200710024763 CN 200710024763 A CN200710024763 A CN 200710024763A CN 101082569 A CN101082569 A CN 101082569A
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- 239000003153 chemical reaction reagent Substances 0.000 title claims abstract description 72
- 150000001413 amino acids Chemical class 0.000 title claims abstract description 63
- 238000000034 method Methods 0.000 title abstract description 20
- 238000006243 chemical reaction Methods 0.000 claims abstract description 20
- 239000005515 coenzyme Substances 0.000 claims abstract description 16
- 108010084238 NAD+ peroxidase Proteins 0.000 claims abstract description 15
- 238000002835 absorbance Methods 0.000 claims abstract description 13
- 238000003556 assay Methods 0.000 claims abstract description 7
- 239000003795 chemical substances by application Substances 0.000 claims description 16
- 102000004316 Oxidoreductases Human genes 0.000 claims description 13
- 108090000854 Oxidoreductases Proteins 0.000 claims description 13
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 claims description 12
- DNIAPMSPPWPWGF-UHFFFAOYSA-N Propylene glycol Chemical compound CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 claims description 12
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 11
- 239000007788 liquid Substances 0.000 claims description 8
- 239000003381 stabilizer Substances 0.000 claims description 7
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 claims description 6
- 235000011187 glycerol Nutrition 0.000 claims description 5
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 4
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 4
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 4
- ACFIXJIJDZMPPO-NNYOXOHSSA-N NADPH Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](OP(O)(O)=O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 ACFIXJIJDZMPPO-NNYOXOHSSA-N 0.000 claims description 3
- 238000013016 damping Methods 0.000 claims description 3
- 239000012530 fluid Substances 0.000 claims description 3
- BOPGDPNILDQYTO-NNYOXOHSSA-N nicotinamide-adenine dinucleotide Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 BOPGDPNILDQYTO-NNYOXOHSSA-N 0.000 claims description 3
- 235000013772 propylene glycol Nutrition 0.000 claims 1
- 238000012360 testing method Methods 0.000 abstract description 14
- 238000010168 coupling process Methods 0.000 abstract description 8
- 238000004737 colorimetric analysis Methods 0.000 abstract description 7
- 102000004190 Enzymes Human genes 0.000 abstract description 5
- 108090000790 Enzymes Proteins 0.000 abstract description 5
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 abstract description 4
- 238000010521 absorption reaction Methods 0.000 abstract description 4
- 238000003745 diagnosis Methods 0.000 abstract description 4
- 230000000694 effects Effects 0.000 abstract description 4
- 230000002255 enzymatic effect Effects 0.000 abstract description 3
- 230000008878 coupling Effects 0.000 abstract description 2
- 238000005859 coupling reaction Methods 0.000 abstract description 2
- 238000006911 enzymatic reaction Methods 0.000 abstract description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 abstract 2
- 230000001590 oxidative effect Effects 0.000 abstract 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 14
- 239000000203 mixture Substances 0.000 description 10
- 238000001514 detection method Methods 0.000 description 7
- 229910052757 nitrogen Inorganic materials 0.000 description 7
- NQMRYBIKMRVZLB-UHFFFAOYSA-N methylamine hydrochloride Chemical compound [Cl-].[NH3+]C NQMRYBIKMRVZLB-UHFFFAOYSA-N 0.000 description 6
- 235000013305 food Nutrition 0.000 description 5
- 210000002700 urine Anatomy 0.000 description 5
- 239000000463 material Substances 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 238000005259 measurement Methods 0.000 description 3
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- 239000000843 powder Substances 0.000 description 3
- 239000000376 reactant Substances 0.000 description 3
- 230000035484 reaction time Effects 0.000 description 3
- DFPAKSUCGFBDDF-UHFFFAOYSA-N Nicotinamide Chemical compound NC(=O)C1=CC=CN=C1 DFPAKSUCGFBDDF-UHFFFAOYSA-N 0.000 description 2
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- 230000001154 acute effect Effects 0.000 description 1
- 230000037354 amino acid metabolism Effects 0.000 description 1
- BIGPRXCJEDHCLP-UHFFFAOYSA-N ammonium bisulfate Chemical compound [NH4+].OS([O-])(=O)=O BIGPRXCJEDHCLP-UHFFFAOYSA-N 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000037444 atrophy Effects 0.000 description 1
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- 210000004185 liver Anatomy 0.000 description 1
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- 238000012423 maintenance Methods 0.000 description 1
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- 208000030159 metabolic disease Diseases 0.000 description 1
- 229960003966 nicotinamide Drugs 0.000 description 1
- 235000005152 nicotinamide Nutrition 0.000 description 1
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Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to a kind of assaying method of the density of amino acid adopting the enzyme colorimetric method and coupling method and an ethanol diagnosis reagent box applying the amino acid oxidizing enzyme coupling NADH peroxidase enzymatic reaction continuous monitor method/ratio colorimetric method. The amino acid oxidizing enzyme enzymolysis amino acid reaction generating hydrogen peroxide and then it is reacted by the effect of NADH peroxidase enzymatic at last makes the coenzyme (there is an absorption peak at the site of 340nm) reduced into the reduction coenzyme (there is an absorption peak at the site of 340nm) so we can assay the degree/speed of the fall-way absorbance of the reduction coenzyme at the place of 340nm. It can measure and calculate the density of amino acid by measuring the degree/speed of the ascent absorbance at the place of 340nm. The method has high specificity and it is not polluted by the endogenous and exogenous object and the test result is precise and accurate. The invention can get the array result by the ultraviolet/visible light analytic instrument so it is convenience to extend and apply.
Description
Technical field
The present invention relates in the fields such as medicine, food, environment detection to amino acid concentration, relate in particular to enzymic colorimetric and coupling method and measure the method for amino acid concentration, and the amino acid diagnose reagent kit that makes thus, belong to the amino acid concentration technical field of analysis and detection.
Background technology
Being determined in medical science/food/industry/agricultural/environment of amino acid content all is important mensuration project.Existing assay method has methods such as chromatography, electrochemical process, instrumental method (amino-acid analyzer), spectrophotometer, operates comparatively numerous and diverse, poor specificity, the instrument and equipment cost is higher.
Amino acid is not simple a kind of material, (instrument costs an arm and a leg can directly to determine 17 seed amino acids with amino-acid analyzer, can not generally use), in medical science/food/industry/agricultural/environment, in most cases, all be that a variety of amino acid exist simultaneously, so need to measure total amino acid content, they can not be represented with the amino acid percent, can only represent with the percent of nitrogen contained in the amino acid (amino acid nitrogen).
Amino acid nitrogen increase in urine when a large amount of food meat or hunger, pregnant woman and neonatal urine amino acid nitrogen also increase.Amino acid metabolism is unusual, causes some amino acid to accumulate in vivo too much, makes that amino acid nitrogen increases in the urine; Acute liver atrophy, liver failure, Reye syndrome or some factor cause protein to decompose the disease of quickening, and the metabolic disorder that genetic disease caused all can make amino acid nitrogen increase in the urine.
Amino acid nitrogen content has specificity height, method is easy, cost is low characteristics in enzymatic assays blood, urine, body fluid, food, soil, the industrial products.
Summary of the invention
The purpose of this invention is to provide a kind of method of measuring amino acid concentration, and use the formulated amino acid diagnose reagent kit of this method.
For realizing purpose of the present invention, a kind of enzymic colorimetric (Enzymatic Colorimetric Method) and coupling method (Couple Reaction) are measured the method for amino acid concentration, utilize amino acid oxidase to be the colour developing enzyme for effect enzyme, NADH peroxidase, amino acid in the testing sample is detected, adopts following steps to carry out:
With sample of measuring and the reagent mixing that contains amino acid oxidase, oxygen, reduced coenzyme, NADH peroxidase, make it to take place following reaction,
The end reaction thing is placed under ultraviolet analyser or half, the automatic clinical chemistry analyzer, detect degree/speed that predominant wavelength 340nm absorbance descends, calculate the size of amino acid concentration.
Above-mentioned enzymic colorimetric and coupling method are measured in the method for amino acid concentration, and described reduced coenzyme is a kind of among NADH, NADPH, thio-NADH or the thio-NADPH.
Realize that amino acid diagnose reagent kit of the present invention can be single agent, is grouped into by following one-tenth:
Damping fluid 20～500mmol/L,
Stabilizing agent 1～4000mmol/L,
Reduced coenzyme 0.1～0.35mmol/L,
Amino acid oxidase 1000～80000U/L,
NADH peroxidase 1000～80000U/L.
Kit can be a dry powder, and use the back that is dissolved in water before use; Also can be mixed with liquid reagent, directly use.
Also the various compositions in above single agent can be carried out formulated in combination and become two agent, such as:
The prescription of two agent is not limited only in the above-mentioned table listed, wherein the composition of reagent I: reduced coenzyme can be placed on reagent II; Amino acid oxidase among the reagent II, NADH peroxidase also can be put into reagent I, so can form multiple formulations, enumerate no longer one by one.Kit can be a dry powder, and use the back that is dissolved in water before use; Also can be mixed with liquid reagent, directly use.
In addition, more than add stabilizing agent usually in the middle of the reagent I/ reagent II of single agent, two agent, concentration is within 1～4000 mmol/L or 0.1%～100% volume ratio scope.
Reagent can also be made into following three reagent:
Similar with two agent, three doses prescription also is not limited only to above-mentioned prescription, wherein the reduced coenzyme among the reagent I can be placed among reagent II or the reagent III, NADH peroxidase among the reagent II can be placed among reagent I or the reagent III, amino acid oxidase among the reagent III also can be put among reagent I or the reagent II, so can form multiple formulations, enumerate no longer one by one.Kit can be a dry powder, and use the back that is dissolved in water before use; Also can be mixed with liquid reagent, directly use.
In addition, for reducing the cross influence between each reagent composition, the stability of maintenance reagent, so that standing storage, usually add stabilizing agent in the middle of the reagent I/ reagent II of above single agent, two agent, three doses the reagent I/ reagent II/ reagent III, active concentration is within 1～4000 mmol/L or 0.1%～100% volume ratio scope.
Material with used as stabilizers can be: at least a in ammonium sulfate (Ammonia Sulfate), glycerine (Glycerol), propylene glycol (Propylene Glycol), the ethylene glycol (Ethylene glycol).
No matter studies show that, take all factors into consideration from the accuracy of measurement result and economy two aspects of preparation cost, be single agent or two agent, and the diagnosis/detection kit of following formula components relation is comparatively desirable, also is preferred version of the present invention:
Damping fluid 100mmol/L,
Stabilizing agent 500mmol/L,
Reduced coenzyme 0.25mmol/L,
Amino acid oxidase 10000U/L,
NADH peroxidase 16000U/L.
Utilize enzymic colorimetric (Enzymatic Colorimetric Method) and coupling method (CoupleReaction) technology, monitoring reduced form nicotinamide coenzyme (reduced coenzyme) is in the variation of 340nm wavelength place absorbance, measured the method for amino acid concentration, simultaneously, the present invention gives in order to realize the amino acid diagnosis/mensuration kit of this method, adopt this reagent not only can be ultraviolet analyser or half, carry out amino acid concentration measurement on the automatic clinical chemistry analyzer, and finding speed is fast, the accuracy height, thereby can obtain practical applying.
The outstanding substantive distinguishing features and the obvious improvement of technical solution of the present invention mainly shows:
(1) the present invention utilizes enzymic colorimetric and coupling method to measure amino acid concentration, and test result is accurate;
(2) composition of participation reaction all adds, and is not subjected to the pollution of inside and outside source material, test process degree of accuracy height;
(3) this method is easy, easy to operate, can obtain testing result fast, and the reaction be under buffer conditions, to carry out, do not pollute the environment;
(4) but this method just fast detecting on general ultraviolet/visible light analysis instrument or semi-automatic/automatic clinical chemistry analyzer does not need special or additional instruments, testing cost is cheap, is convenient to apply in the industry;
(5) use the reagent that assay method provided by the invention can be made various ways such as liquid reagent, powdered reagent, be used for measuring the size of various sample amino acid concentrations;
(6) liquid amino acid diagnosis/detection kit provided by the invention, good stability has guaranteed the application testing effect well.Be made into after two agent, can further reduce the cross influence between the various compositions, testing result is more credible, and reagent is more stable, can store for a long time.
Embodiment
The assay method of a kind of amino acid concentration of the present invention and amino acid diagnose reagent kit, utilization amino acid oxidase (amino acid oxidase; EC 1.4.3.2; EC 1.4.3.3) coupling NADH peroxidase (NADH peroxidase; EC 1.11.1.1; EC 1.11.1.2) enzymatic reaction continuous monitoring method/speed ratio color method.The reaction of amino acid oxidase enzymolysis amino acid produces hydrogen peroxide, the effect of uniting the NADH peroxidase again by (idol), reduced coenzyme (absorption peak being arranged at the 340nm place) is oxidized into coenzyme (not having absorption peak at the 340nm place) the most at last, thereby measured degree/speed that reduced coenzyme descends in 340nm place absorbance, by measuring degree/speed that 340nm place absorbance descends, can calculate amino acid whose concentration.
Below in conjunction with specific embodiment technical solution of the present invention is described further.These examples only are some exemplary applications, can not be interpreted as a kind of restriction to claim protection domain of the present invention.
Embodiment one (single agent)
Prepare amino acid diagnose reagent kit by following composition and consumption:
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L,
Ammonium sulfate 500mmol/L,
NADH 0.25mmol/L，
Amino acid oxidase 10000U/L,
NADH peroxidase 16000U/L;
Reagent divides the bottle of packing into after all dissolving and preparing, and carries out freeze drying, makes powdered reagent; Before the use, add pure water, use after redissolving.
On automatic clinical chemistry analyzer, set: 37 ℃ of temperature, 10 minutes reaction time, initial absorbance 1.8 ± 0.7, test predominant wavelength 340nm, test commplementary wave length 405nm, the volume ratio of tested amino acid sample and reagent is 1: 25, and the Direction of Reaction is negative reaction (reaction descends), about 5 minutes detection times.
After adding sample and reagent, make them mixed and have reaction, reactant places under the Biochemical Analyzer the most at last, detects degree/speed that predominant wavelength 340nm absorbance descends, thereby calculates amino acid whose concentration.
Embodiment two (two agent)
Prepare amino acid diagnose reagent kit by following composition and consumption:
Reagent I---
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L,
Ammonium sulfate 50mmol/L,
NADPH 0.25mmol/L；
Reagent II---
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L,
Glycerine 500mmol/L,
Amino acid oxidase 10000U/L,
NADH peroxidase 16000U/L.
Reagent divides the bottle of packing into after all dissolving and preparing, and makes liquid double reagent, can directly use.
On automatic clinical chemistry analyzer, set: 37 ℃ of temperature, 10 minutes reaction time, initial absorbance 1.8 ± 0.7, test predominant wavelength 340nm, test commplementary wave length 405nm, the volume ratio of tested amino acid sample and reagent I, reagent II is 2: 20: 5, and the Direction of Reaction is negative reaction (reaction descends), about 5 minutes detection times.
After adding sample and reagent, make them mixed and have reaction, reactant places under the Biochemical Analyzer the most at last, detects degree/speed that predominant wavelength 340nm absorbance descends, thereby calculates amino acid whose concentration.
Embodiment three (three doses)
Prepare amino acid diagnose reagent kit by following composition and consumption:
Reagent I---
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L,
Propylene glycol 50mmol/L,
thio-NADH 0.25mmol/L；
Reagent II---
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L,
Ethylene glycol 500mmol/L,
NADH peroxidase 16000U/L;
Reagent III---
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L,
Glycerine 500mmol/L,
Amino acid oxidase 10000U/L;
Reagent divides the bottle of packing into after all dissolving and preparing, and makes liquid three reagent, can directly use.
When measuring amino acid concentration, on automatic clinical chemistry analyzer, set: 37 ℃ of temperature, 10 minutes reaction time, initial absorbance 1.8 ± 0.7, test predominant wavelength 340nm, test commplementary wave length 405nm, the volume ratio of tested amino acid sample and reagent I, reagent II, reagent III is 4: 40: 5: 5, the Direction of Reaction is negative reaction (reaction descends), about 5 minutes detection times.
After adding sample and reagent, make them mixed and have reaction, reactant places under the Biochemical Analyzer the most at last, detects degree/speed that predominant wavelength 340nm absorbance descends, thereby calculates amino acid whose concentration.
The applicant adopts other various reduced form chromogens combinations of putting down in writing in the above summary of the invention all can reach purpose of the present invention through experimental verification, in view of situation such as determination step and above embodiment roughly the same, do not separately enumerate.
In a word, experiment showed, and adopt assay method of the present invention can draw required measurement result by general biochemical analyzer fully, and highly sensitive, degree of accuracy good, be not subjected in the pollution of allogenic material, easy to utilize.
Claims (6)
1. the assay method of amino acid concentration is characterized in that may further comprise the steps:
3) the end reaction thing is placed under ultraviolet analyser or half, the automatic clinical chemistry analyzer, detect degree/speed that predominant wavelength 340nm absorbance descends, calculate the size of amino acid concentration.
2. amino acid diagnose reagent kit, reagent is grouped into by following one-tenth in the box:
Damping fluid 20～500mmol/L,
Stabilizing agent 1～4000mmol/L,
Reduced coenzyme 0.1～0.35mmol/L,
Amino acid oxidase 1000～80000U/L,
NADH peroxidase 1000～80000U/L.
3. amino acid diagnose reagent kit according to claim 2 is characterized in that: described stabilizing agent is at least a in ammonium sulfate, glycerine, propylene glycol, the ethylene glycol.
4. amino acid diagnose reagent kit according to claim 2 is characterized in that: described reduced coenzyme is a kind of among NADH, NADPH, thio-NADH or the thio-NADPH.
5. any one amino acid diagnose reagent kit in the claim 2～4 is characterized in that: described reagent is made into single agent or two agent or three doses.
6. any one amino acid diagnose reagent kit in the claim 2～4, it is characterized in that: described kit is powdered reagent box or liquid reagent box.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200710024763 CN101082569A (en) | 2007-06-28 | 2007-06-28 | Method for measuring amino acid concentration and amino acid diagnose reagent kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200710024763 CN101082569A (en) | 2007-06-28 | 2007-06-28 | Method for measuring amino acid concentration and amino acid diagnose reagent kit |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101082569A true CN101082569A (en) | 2007-12-05 |
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| Application Number | Title | Priority Date | Filing Date |
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| CN 200710024763 Pending CN101082569A (en) | 2007-06-28 | 2007-06-28 | Method for measuring amino acid concentration and amino acid diagnose reagent kit |
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| Country | Link |
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| CN (1) | CN101082569A (en) |
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2007
- 2007-06-28 CN CN 200710024763 patent/CN101082569A/en active Pending
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